Biologically active catecholamine derivatives

ABSTRACT

Biologically active derivatives of norepinephrine are disclosed. Such derivatives are catecholamines wherein isoproterenol is modified by extending the isopropyl functional grouping to an alkyl, aryl, or alkyl-aryl chain of variable length where said chain terminates in a carboxylic acid functional group, or in a substituted amide functional group. Such derivatives are β-adrenergic and a number of such derivatives e.g., 6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid p-toluide, 6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid para-n-butyl anilide; 6-(β,3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid para-methoxy anilide; 6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid paratrifluoromethyl anilide; and 6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid N-methyl para-toluide display a β-adrenergic activity at least several orders of magnitude greater than isoproterenol itself. Several methods of producing such active derivatives are also disclosed.

DESCRIPTION BACKGROUND OF THE INVENTION

The role of the hormone norepinephrine,4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, and its congeners such asepinephrine or isoproterenol as transmitter substances of the peripheralsympathetic nerve endings and of certain synapses in the central nervoussystem is well documented. Epinephrine,4-[1-hydroxy-2-(methyamino)-ethyl]-1,2-benzenediol, which acts as astimulator of the sympathetic nervous system, thus producing a broadrange of physiological effects such as vasopression, increased bloodpressure, cardiac stimulation, increased cardiac output, glucose releaseand glycogenolysis, is also extremely well known and studied.Generically in a chemical sense, such hormones are classified ascatecholamines. That is, they possess the characteristic hydroxyl groupssubstituted at the 3 and 4 positions on a benzene ring and thehydroxy-alkylamine side chain attached to the number 1 carbon of thebenzene ring. More specifically norepinephrine has the structure:##STR1## while epinephrine has the structure: ##STR2##

Although norepinephrine and epinephrine are closely related chemically,their physiological effects are somewhat different with respect toreactions mediated by the hormones, tissue and organs affected, and thestrength of their respective activities. Thus, norepinephrine primarilymediates nerve impulses as a transmitter substance of the sympatheticnerve endings. Epinephrine, on the other hand, operates primarily as avasopressor, heart stimulant, and blood volume and pressure stimulant.The two molecular structures norepinephrine and epinephrine producequalitatively similar but quantitatively different physiologicaleffects. In some instances these effects are diverse and general,whereas, in others, the effect is very specific. It is therefore ofinterest to explore the possibility of devising epinephrine andnorepinephrine related molecules which will exhibit biological activityin general and perhaps selected activity which will permit "targeting"various physiological functions on a more selective basis than ispossible with the naturally occurring hormones.

Unfortunately, the epinephrine and norepinephrine structures,(generically-catecholamines) are particularly sensitive to alteration ofthe molecular structure insofar as maintenance of physiological activityis concerned. Thus when aromatic substitutions are made at the 2 or 5carbon positions on the benzene ring, activity may be destroyed. On theother hand, a number of active molecules have been obtained byalkylation of the side chain amine group, e.g., the isopropyl homolog ofepinephrine, isoproterenol; which has the structure: ##STR3##

The preparation of other biologically active catecholamines would, ofcourse, be of great interest, especially where such derivatives mayexhibit specialized effects on biological systems, or where thederivative could be further conjugated with other "carrier" molecularstructures without affecting the basic physiological functions which arecharacteristic of the catecholamine hormones.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to modified catecholamine hormoneswherein isoproterenol is chemically modified by extending the side chainisopropyl group to add further an alkyl or alkyl-aryl chain of variablelength to terminate in a carboxylic acid group or carboxylic acidderivatives such as an amide or substituted amide. Such modifiedcatecholamine hormones have the general structure: ##STR4##

In order to prevent self-degradation upon storage, the modifiedcatecholamines are usually prepared in the protonated form, i.e.:##STR5## wherein X.sup.⊖ may be any pharmaceutically acceptable anionsuch as chloride, acetate, sulfate, phosphate and the like.

It is an object of the invention to provide biologically activederivatives of norepinephrine or isoproterenol.

It is another object of the invention to provide isoproterenolderivatives which exhibit β-adrenergic activity.

It is still another object of the invention to provide β-adrenergicallyactive derivatives of isoproterenol wherein the catecholamine ismodified by extending the isopropyl group to an alkyl chain of variablelength and terminating in a carboxylic acid group or a substituted amidegroup.

It is a further object of the invention to prepare β-adrenergicallyactive derivatives of isoproterenol having the general formula: ##STR6##wherein n is 1 to 15 and --CO--R is a carboxylic acid group or acarboxylic acid derivative such as an amide or substituted amide.

Other objects and advantages of the invention will be apparent from thefollowing description and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to derivatives of isproterenol whichexhibit biological activity; and more specifically to isoproterenolderivatives wherein isoproterenol is modified by the addition of alkylor alkyl-aryl chains of variable length to the side chain aminofunctional group. Such added chains are further characterized by theirtermination in a carboxyl functional group or in a derivative of acarboxylic acid such as an amide in a substituted amide.

Generically, the biologically active isoproterenol derivatives have thestructure: ##STR7## wherein n indicates the number of methylene groupsin the chain, but which is most usually from 1 to perhaps 15, and R maybe either --OH, i.e., a carboxylic acid termination; or --NH--R', i.e.,an amide termination.

While it is indicated that the added alkyl chain is a straight chain, itshould be understood that the chain may also be branched; and may eveninclude aryl groupings or mixed alkyl-aryl groupings. The importantcriterion is the retention or enhancement of β-adrenergic activity. Inany event, however, the straight chain alkyls i.e., --(CH₂)_(n) -- doappear to favor the retention of the β-adrenergic activity.

It is of equal importance that the added N substituent groupingterminates in a carboxyl functional group, i.e., --COOH; or in an amide,i.e., --CO--NH--R' generally where R' can be H (a primary amide) or avariety of alkyl or aryl groups as outlined below. Such terminal amidesmay be derived from aromatic amines such as aniline and anilinederivatives such as para-toluidine, ##STR8## which is especiallypreferred; para-n-butyl aniline, ##STR9## p-methoxy aniline, ##STR10##p-trifluoromethyl aniline, ##STR11## n-methyl p-toluidine, ##STR12## Theterminal amide group may also be derived from alkyl amines, e.g.,n-butyl amine

    --NH--(CH.sub.2).sub.3 --CH.sub.3 ;

or similar straight or branch chain amines or cycloalkyl amines e.g.,cyclohexyl amine, ##STR13## or the like.

As noted previously, inspection of the structure of the biologicallyactive norepinephrine derivatives will reveal that there is a side-chainmethyl group attached to the carbon immediately adjacent the side chainamine. Since isoproterenol has the structure: ##STR14## it will beapparent that the compounds of the invention may also be characterizedas isoproterenol derivatives. That is, the invention compounds may becharacterized as modifications of isoproterenol wherein the terminalisopropyl group is extended to an alkyl chain, or alkyl-aryl chain ofvariable length wherein the chain terminates in a carboxylic acid orsubstituted amide.

Such modified and extended isoproterenol derivatives have demonstratedβ-adrenergic activity in vitro by measuring cyclic AMP (cAMP) formationin S49 mouse lymphoma cells, and in vivo activity by monitoring changesin the blood pressure and heart rate of rats. The in vitro testing ofcAMP formation in S49 lymphoma cells is a well documented and acceptedtest for indicating and anticipating β-adrenergic activity in themammalian body.

Although the general structure of the invention derivatives was notedabove in the "amine" form, i.e., ##STR15## from a practical standpoint,the compounds are normally prepared in the protonated form, i.e.,:##STR16## wherein X.sup.⊖ may be any pharmaceutically acceptable anionsuch as acetate, chloride, sulfate, phosphate etc. The protonated saltform of the derivative exhibits far greater stability in storage thanthe "amine" form which undergoes internal degradation reactions, thusdestroying its activity.

Methods for Synthesizing the Derivatives

The carboxylic acid terminated isoproterenol derivatives may besynthesized by a reductive amination reaction between norepinephrine andthe appropriate keto-acid. The reductions are performed in the presenceof platinum oxide (PtO₂) catalyst at room temperature and at atmosphericpressure under hydrogen. The reaction is carried out in methanol or inmixtures of methanol and acetic acid.

The reaction scheme (Route A) is: ##STR17##

The substituted amide terminated norepinephrine derivatives may besynthesized in some instances, by carbodiimide coupling of thecarboxylic acid terminated derivative. The carbodiimide coupling agentmay be water soluble, such as 1-ethyl-3(3dimethyl-aminopropyl)carbodiimide hydrochloride; or water insoluble,such as dicyclohexyl carbodiimide, depending on the solubility of theamine to which the carboxylic acid is being coupled. Thus a carboxylicacid terminated derivative as prepared in the reaction scheme notedabove, may, in turn, be converted into the substituted amide by reactingthe carboxylic acid derivative with the desired substituted amine in thepresence of carbodiimide.

The reaction scheme is (Route A): ##STR18##

The carbodiimide reaction path is generally suitable only for chainlengths where n=4 or higher. Shorter members of the series tend to formlactams under the coupling conditions.

Therefore, a more general synthesis scheme (Route B) for the productionof the substituted amide derivatives is the reductive amination of theappropriate ketoamide with norepinephrine.

The appropriate ketoamide may be prepared from the keto acid by reactionwith the appropriate amine and a coupling agent such as a carbodiimide.The keto acids, in turn, can be previously prepared in accordance withmethods well known in the prior art.

The general synthesis of the amides via Route B is: ##STR19##

Synthesis of Representative Compounds

All of the catecholamine derivatives can be synthesized by one ofseveral general routes as noted above. For clearer understandingspecific examples are described below. From a review of these examples,the synthesis of all the relevant catecholamines will be apparent.

Synthesis of 6-(β-3,4-Dihydroxyphenyl-β-hydroxy)-ethylamino heptanoicacid p-Toluide

This target molecule was prepared by the two routes A and B, outlinedabove.

Route A δ-Acetyl-n-valeric Acid (see Compound (2) in Route A above wheren=4)

This compound was prepared from 2-methylcyclohexanol according toSchaeffer and Snoddy (Org. Syn., 31, 3 (1951)).

6-(β-3,4-Dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid (seeCompound (3) above where n=4 and, Table 1, Compound 6)

Norepinephrine as the free base (see compound 1 above, 3.91 g, 0.023mmol) and δ-acetyl-n-valeric acid (see Compound (2) above where n=4,7.50 g, 0.046 mmol) were dissolved in 20% acetic acid mixed intomethanol (75 ml). Hydrogenation was performed overnight at roomtemperature and atmospheric pressure over 0.1 g PtO₂ catalyst. Afterfiltration and washing with acetic acid until only platinum blackremained on the filter, the combined filtrate was evaporated. Theresulting thick oil was dissolved in methanol and gradually added toethyl ether. The whitish precipitate was filtered, washed with ether,and dried in vacuo giving 5.06 g of an amorphous solid (compound 6,Table 1, 74% yield), which was homogeneous by TLC and NMR.

The following compound was prepared by two methods using differentcarbodiimide coupling agents:

6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino Heptanoic Acid p-Toluide(Compound 12 in Table 1 below) Method(i)

p-Toluidine hydrochloride was prepared by addition of 4 N HCL/dioxane toa solution of the free base in ether. The crystals were collected byfiltration, washed with ether, and dried in vacuo.

Compound (3), n=4 (2.97 g, 10 mmol), i.e., and p-toluidine hydrochloride(1.86 g, 13 mmol) were added to 0.05 M monosodium phosphate (400 ml)under an inert atmosphere.1-Ethyl-3(3-dimethyl-aminopropyl)-carbodiimide HCl (Sigma, 3.3 g, 17mmol) was added. After one day the solution was extracted withchloroform (four times) and with n-butanol (three times). The combinedbutanol extracts were evaporated under reduced pressure at 40° C. Theresulting oil was redissolved in butanol, filtered, and evaporatedwithout delay. This oil was transformed into a white amorphous solid bystirring under ether. The solid was filtered, washed with ether, anddried in vacuo giving 1.73 g (41% yield) of the hydrochloride salt ofthe title compound (Compound 12, Table 1).

Method (ii)

Compound (3) above, (n=4,102 mg, 2 equivalents) and p-toluidinehydrochloride (25 mg, 1 equivalent) were dissolved in 5 ml ofpyridine/water (80%, v/v). Dicyclohexylcarbodiimide (88 mg, 2.5equivalents) was added and the solution was stirred under nitrogenovernight. Solvent was removed under reduced pressure at 40° C. until awhite precipitate formed. Water (15 ml) was added and the urea wasremoved by filtration through Celite.

The solution was passed over a bed of Bio-Rex 70 cation exchange resin(Bio-Rad, 2 g dry weight) in the H⁺ form. After washing the bedthoroughly with H₂ O, the product was removed from the resin with 5%acetic acid. After lyophilization, there remained 40 mg (51% yield) of awhite solid, the acetate salt of the title compound (Compound 12, Table1, X=CH₃ COO). This was shown by TLC to be identical to the productprepared by method (i) and free from starting materials.

Route B δ-Acetyl-n-valeric Acid p-Toluide (see Compound (4) above wheren=4)

δ-Acetyl-n-valeric acid (1.2 g) and p-toluidine (1.0 g) were dissolvedin tetrahydrofuran (30 ml). Dicyclohexylcarbodiimide was added and thesolution stirred overnight. The solvent was removed, and the residuesuspended in ethyl acetate and filtered. After washing with 0.1 Nhydrochloric acid, saturated sodium bicarbonate, and water, the organiclayer was dried over magnesium sulfate. The solution was filtered andevaporated. Recrystallization of the residue from ethyl acetate/hexanegave 1.36 g (70% yield) of white crystals. The δ-acetyl-n-valeric acidp-toluide (see Compound (4) above) so produced had a m.p. of 106°-107.5°C. Analysis calculated for C₁₄ H₁₉ NO₂ : C, 72.07; H, 8.21; N, 6.00.Found: C, 71.86; H, 8.28; N, 6.04.

Norepinephrine, free base (1, 30 mg, 0.16 mmol) and Compound (4) above(n=4, 74.5 mg, 0.32 mmol) were dissolved in acetic acid (1 ml). PtO₂catalyst (10 mg) was added and the mixture was hydrogenated atatmospheric pressure overnight. The solution was removed from thecatalyst and added to 0.01 N hydrochloric acid, after which extractionswith ethyl acetate and n-butanol were performed. The combined butanolextracts were evaporated, redissolved in n-butanol, filtered through aglass wool plug in a Pasteur pipette, and evaporated. The oil was driedin vacuo giving 62 mg of the HCl salt of the title compound (Compound12, Table 1, X=Cl).

Biological Activity

The following Table 1 sets forth the results of the testing ofbiological activity of the indicated norephinephrine derivatives.

The activities, i.e., K_(A) and E_(max), were observed in tests of thenoted catecholamines on S49 lymphoma cells, which is utilized as ameasure of agonist activity. Such testing procedures and theirsignificance has been set forth in a number of prior publications, e.g.Shear et al., J. Biol. Chem.; 251, 7572 (1976); Coffino, et al., InVitro, 14, No. 1, 140 et.seq. (1978); and in Johnson, et al.; MolecularPharmacology, 15, 16-27 (1978). The tests measure increased cyclicadenosine monophosphate (cAMP) concentration in the S49 cells. In Table1, K_(A) indicates the concentration of the catecholamine forapproximately half maximal stimulation of cAMP concentration. E_(max) isa measure of the maximal response (cAMP concentration) of the S49 cellsto the respective catecholamines regardless of how high theconcentration of the catecholamines may become. The Table compares theresponse of the cells to the particular catecholamine derivatives withthe cells' response to isoproterenol. Thus a measure of the relativebiological activity can be obtained.

                                      TABLE 1                                     __________________________________________________________________________    Biological Activity of Catecholamine Derivatives                               ##STR20##                                                                                                         d,l-Isoproterenol                        Compound                                                                            R           n Synthesis.sup.a                                                                      K.sub.A (M)                                                                          E.sub.max                                                                        K.sub.A (M)                                                                          E.sub.max                         __________________________________________________________________________     6    OH          2 Route B                                                                              0.16 × 10.sup.-5                                                               273                                                                              0.25 × 10.sup.-9                                                               366                                7    OH          3 Route B                                                                              0.37 × 10.sup.-6                                                               254                                                                              0.25 × 10.sup.-9                                                               366                                8    OH          4 Route B                                                                              0.35 × 10.sup.-6                                                               282                                                                              0.34 × 10.sup.-9                                                               414                                9    OH          5 Route B                                                                              0.35 × 10.sup.-6                                                               170                                                                              0.29 × 10.sup.-9                                                               248                               10                                                                                   ##STR21##  2 Route B                                                                              0.35 × 10.sup.-8                                                               147                                                                              0.25 × 10.sup.-8                                                               186                               11                                                                                   ##STR22##  3 Route B                                                                              0.80 × 10.sup.-8                                                               171                                                                              0.25 × 10.sup.-8                                                               186                               12                                                                                   ##STR23##  4 Routes A, B                                                                          0.56 × 10.sup.-10                                                              201                                                                              0.25 × 10.sup.-8                                                               186                               .sup. 13.sup.b                                                                       ##STR24##  4 Route B                                                                              0.60 × 10.sup.-11                                                              269                                                                              0.43 × 10.sup.-10                                                              341                               14                                                                                   ##STR25##  5 Route A                                                                              0.22 × 10.sup.-7                                                               195                                                                              0.25 × 10.sup.-8                                                               186                               15                                                                                   ##STR26##  4 Route B                                                                              0.59 × 10.sup.-15                                                              151                                                                              0.77 × 10.sup.-12                                                              124                               16                                                                                   ##STR27##  4 Route B                                                                              0.13 × 10.sup.-7                                                               166                                                                              0.40 × 10.sup.-7                                                               136                               17                                                                                   ##STR28##  4 Route B                                                                              < 10.sup.-15                                                                         150                                                                              0.77 × 10.sup.-12                                                              124                               18                                                                                   ##STR29##  4 Route B                                                                              0.22 × 10.sup.-13                                                              206                                                                              0.16 × 10.sup.-11                                                              192                               19    NH(CH.sub.2).sub.3CH.sub.3                                                                4 Routes A, B                                                                          0.18 × 10.sup.-9                                                               229                                                                              0.45 × 10.sup.-10                                                              318                               20                                                                                   ##STR30##  4 Routes A, B                                                                          0.26 × 10.sup.-9                                                               245                                                                              0.45 × 10.sup.-10                                                              318                               __________________________________________________________________________     .sup.a Refer to description                                                   .sup.b from Lnorepinephrine                                              

A large dependence of activity on the length of the methylene chaini.e., (CH₂)_(n) is seen with the toluides (Compounds 10-14). Of thecompounds noted, peak activity is seen in the case of n=4 (Compound 12),which has an activity approximately two orders of magnitude greater thanthat of isoproterenol. These results are supported by data from rattesting, which have shown that Compound 12 is 50-70 times more activethan isoproterenol, when blood pressure and heart rate changes aremonitored on administration of the drug. Furthermore, the K_(D) 's forcompound 12 (the concentration of drug at which half the receptors arenot occupied) in S49 and C6 cells are 2 orders of magnitude lower thanwould be found for isoproterenol.

Similarly, there is a dependence on the nature of the substituent groupon the amide, aromatic amides (Compounds 10-18) being considerably morebiologically active than aliphatic amides (Compounds 19-20) in thistest. The nature of substituent groups on the ring of aromatic amidesalso appears to exert a profound effect on biological activity in thesecompounds. Thus Compound 16, for example, which contains a methoxylgroup on the ring has approximately the same activity as isoproterenolin the S49 cell assay. Compound 12, on the other hand, which has amethyl group at the para position on the ring is almost 2 orders ofmagnitude more active than isoproterenol; and Compounds 15 and 17 whichcontain n-butyl and trifluoromethyl substituents, respectively, are atleast 3 orders of magnitude more active than isoproterenol. Thus it canbe expected that elaboration of the substituent groups on the aromaticring will lead to a wide variety of β-adrenergic drugs.

We claim:
 1. β-adrenergic compounds having the formula: ##STR31##wherein n=1 to 15, and R is NHR', where R' is H or an alkyl, aryl, oralkyl-aryl substituent.
 2. β-adrenergic compounds in the protonatedform: ##STR32## wherein n=1 to 15, R is NHR', where R' is an H or alkyl,aryl, or alkyl-aryl substituent and X.sup.⊖ is a pharmaceuticallyacceptable anion.
 3. The compounds of claim 1 wherein n=1 to
 5. 4. Thecompounds of claim 1 wherein n=4.
 5. β-adrenergic compounds having theformula: ##STR33## wherein n=1 to 15, and R is an aniline derivative. 6.The compounds of claim 5 wherein n=4.
 7. The compounds of claim 5wherein the aniline derivative is ##STR34##
 8. The compounds of claim 7wherein n=4.
 9. The compounds of claim 5 wherein the aniline derivativeis ##STR35##
 10. The compounds of claim 9 wherein n=4.
 11. The compoundsof claim 5 wherein the aniline derivative is ##STR36##
 12. The compoundsof claim 11 wherein n=4.
 13. The compounds of claim 5 wherein theaniline derivative is ##STR37##
 14. The compounds of claim 13 whereinn=4.
 15. The compounds of claim 5 wherein the aniline derivative is:##STR38##
 16. The compounds of claim 15 wherein n=4.
 17. β-adrenergiccompounds having the formula: ##STR39## wherein n=1 to 15, and R is analkyl amine.
 18. The compounds of claim 17 wherein n=4.
 19. Thecompounds of claim 17 wherein the alkyl amine is --NH(CH₂)₃ --CH₃. 20.The compounds of claim 19 wherein n=4.
 21. β-adrenergic compounds havingthe formula: ##STR40## wherein n=1 to 15, and R is a cyclo alkyl amine.22. The compounds of claim 21 wherein n=4.
 23. The compounds of claim 21wherein the cyclo alkyl amine is ##STR41##
 24. The compounds of claim 23wherein n=4.
 25. The compound 6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid p-toluide andpharmaceutically acceptable salts thereof.
 26. The compound6-(β-3,4-dihydroxy phenyl-β-hydroxy)-ethylamino heptanoic acidpara-n-butyl anilide and pharmaceutically acceptable salts thereof. 27.The compound 6-(β-3,4-dihydroxy phenyl-β-hydroxy)-ethylamino heptanoicacid para-methoxy anilide and pharmaceutically acceptable salts thereof.28. The compound 6-(β-3,4 dihydroxy phenyl-β-hydroxy)-ethylaminoheptanoic acid para-trifluoromethyl anilide and pharmaceuticallyacceptable salts thereof.
 29. The compound 6-(β-3,4-dihydroxyphenyl-β-hydroxy)-ethylamino heptanoic acid N-methyl para-toluide andpharmaceutically acceptable salts thereof.